Can someone advise on collecting sap from frozen xylem vessels for metabolomics?

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Guido Bongi

Each tissue has got its degrading enzymes from tubers to stem tissues wich are alive after twaving. I think you can measure the oxygen uptake, dephosphorylation or solution phenolics oxydations with twaving in action and then decide what is the case. Then you can try combinations of reductants like 2ME or more specific inhibitors like phenylmethyl sulphonyl fluoride (PMSF) or sacrifical proteins like serum albumine, or use trichloroactetic acid TCA, but all depends on what type of metabolite you want to be stable.

Ruzica Apostolova

I think that "defrosting procedure the enzymatic activity might effect the sample's metabolomic profile".. Is ok

Noam Reshef

Ruzica, could you please explain your answer?

Thank you Guido. There is a very wide range of metabolites that I'm interested in, from hormones to phenols and primary metabolites (metabolomics). In addition the samples might be used for miRNA analysis. As you can understand, there is a very wide range of metabolites that I want to protect from possible degradation. What is 2ME that you specified ?

Phillip: From field grown vines I cut shoots at about 30cm down from the apex. With a small knife I strip out the phloem tissue (which goes out very clean and easily on green, fresh shoots) and cut the remaining xylem (with pith) to 3-5cm pieces, wrap in aluminum foil and flash freeze it in liquid Nitrogen. The whole process takes about 1min.

Thank you Phillip.

The only contaminating live cells left in the samples are cells from the pith. A comparison between the centrifuge and a direct extract might reveal whether the centrifuge takes out the sap with minimal damage to the tissue cells. Using fresh tissues is a last resort since the process will take a long time and will probably lead to the degradation of sensitive metabolites as ABA.